Preclinical gene therapy studies for hemophilia using adenoviral vectors

Hemophilia A and B are hereditary coagulation defects resulting from a deficiency of factor VIII (FVIII) and factor IX (FIX), respectively. Introducing a functional FVIII or FIX gene could potentially provide a cure for these bleeding disorders. Adenoviral vectors have been used as tools to introduce potentially therapeutic genes into mammalian cells and are by far the most efficient vectors for hepatic gene delivery. Long-term expression of both FVIII and FIX has been achieved in preclinical (hemophilic) mouse models using adenoviral vectors. Therapeutic levels of FVIII and FIX also have been achieved in hemophilic dogs using adenoviral vectors and in some cases expression was long-term. The performance of earlier generation adenoviral vectors, which retained residual viral genes, was compromised by potent acute and chronic inflammatory responses that contributed to significant toxicity and morbidity and short-term expression of FVIII and FIX. The development of improved adenoviral vectors devoid of viral genes (gutless or high-capacity adenoviral vectors) was therefore warranted, which led to a significant reduction in acute and chronic toxicity and more prolonged expression of FVIII and FIX. Strategies aimed at making these vectors safer and less immunogenic and their implications for hemophilia gene therapy are discussed in this review.     

Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.     

Clinical gene transfer studies for hemophilia A

The recent advances in gene transfer technology have expedited the development of gene therapy for the treatment of hemophilia A. Three different U.S. Food and Drug Administration-approved phase I clinical trials had been initiated using different gene therapy approaches each with their own advantages and limitations. In the first gene therapy trial for hemophilia A, a non-viral approach was being explored for patients with severe hemophilia A using ex vivo transfected dermal fibroblast expressing B-domain-deleted factor VIII ( BDD-FVIII). There were no serious adverse events and some patients appeared to have experienced fewer bleeding episodes with very low levels of FVIII near baseline. In the second trial, onco-retroviral vectors expressing BDD-FVIII were injected by peripheral intravenous infusion in adult patients suffering from severe hemophilia A. The procedure was safe and in some patients FVIII-transduced cells were detectable in the peripheral blood for more than a year. Although no sustained FVIII expression was detectable, occasional modest changes in FVIII levels were apparent, and in some cases a reduced bleeding frequency occurred compared with historical rates. In another trial, one patient suffering from severe hemophilia A has been treated with a high-capacity (or gutless) adenoviral vector expressing full-length FVIII, which appeared to have resulted in 1% of normal FVIII levels for several months. However, a transient inflammatory response with hematologic and liver abnormalities was observed. In conclusion, although modest improvements in clinical end points have been detected in some patients in these early phase I trials, further improvements in gene delivery technologies are warranted to bring hemophilia A gene therapy one step closer to reality.     

Clinical gene transfer studies for hemophilia B

Hemophilia B, a deficiency of functional factor IX (FIX), has been extensively explored as a model for gene transfer. Two U.S. Food and Drug Administration-approved clinical studies for hemophilia B have been undertaken, both using adeno-associated viral vectors (AAV). AAV vectors have tropism for liver, muscle, central nervous system, and the respiratory tract; both skeletal muscle and liver have been used as target tissues in the hemophilia B studies. In both studies, proof of principle was first established in the hemophilia B dog model, with long-term expression of canine FIX at therapeutic levels achieved before clinical studies were initiated. In the AAV-FIX muscle trial, vector was introduced into skeletal muscle of the upper and lower extremities of eight human patients by direct intramuscular injection. Muscle biopsies taken 2 to 10 months postinjection demonstrated gene transfer and expression (by Southern blot and immunofluorescence, respectively) in all patients, but circulating FIX levels were generally not >1%, and escalation of dose to levels that proved therapeutic in animals was thwarted by feasibility issues regarding the number of injections required. Nevertheless, the study demonstrated that parenteral injection of AAV-FIX was safe at the doses tested, and could result in long-term expression of the transgene. Moreover, the general characteristics of transduction of human muscle were similar to those observed in other animal models. The safety and efficacy data established in the first trial formed the basis for a second trial in which AAV-FIX is administered systemically to target the liver. The liver study is currently ongoing, with six patients enrolled to date.     

Preclinical gene therapy studies for hemophilia using adeno-associated virus (AAV) vectors

Gene therapy offers a potential cure for hemophilia and several gene transfer vectors have been evaluated for their ability to treat this disease. This article reviews the studies that have been performed to evaluate the ability of recombinant adeno-associated virus (AAV) vectors to achieve safely the sustained expression of clotting factors following intramuscular, intravenous, and intrahepatic delivery to several animal models. These routes of administration are all effective in providing sustained and therapeutic levels of factor IX (FIX), although the levels vary. Intrahepatic delivery is more efficacious than intravenous administration, which is superior to intramuscular delivery. The recent development of efficient factor VIII (FVIII) expression cassettes has made AAV-based gene therapy for hemophilia A also within reach. Although no acute toxicity has been observed with any route of administration, an increased risk of antibody formation against FIX has been noted following intramuscular delivery. Biodistribution studies concluded that the vector disseminates to most tissues in a dose-dependent and time-dependent manner, but the majority of the vector resides in the targeted tissue. In addition, the risk of germline transmission has been shown to be low or absent. The relatively recent isolation of new AAV serotypes has resulted in the identification of vectors that have enhanced tropism for certain tissues. This combined with the potential of these new vectors to evade the immune response to AAV2, makes them attractive candidates for gene therapy. Although much progress has been made using AAV to treat hemophilia, there are several outstanding issues that need to be addressed. Delivery of AAV to large animals has not been reproducible, which could be due to nonoptimized delivery and/or immune responses to the vector or transgene product. In addition, a complete understanding of the biology of these vectors is required to assess their long-term safety. Solving these issues will lead to the development of a successful gene therapy product.     

Immune implications of gene therapy for hemophilia

Similar to any novel treatment strategy for hemophilia, gene therapy faces the question of the risk of formation of inhibitory antibodies to the therapeutic factor VIII or factor IX protein. Activation of CD4 (+) or CD8 (+) T cells could lead to antibody formation or cytotoxic T lymphocyte responses to transgene-expressing cells. Preclinical studies in animal models of hemophilia A and B with different mutations in the dysfunctional gene shed light on the risk for such immune responses and point toward strategies to avoid immune activation or even promote tolerance induction. The impacts of variables such as choice and design of gene transfer vector, underlying gene mutation, route of vector administration, and transient immune suppression are discussed. Maintenance of immunological hyporesponsiveness to the therapeutic gene product is critical for successful gene therapy. Recent studies provide evidence for tolerance induction to coagulation factor antigens by viral hepatic or neonatal in vivo gene transfer, by in utero gene delivery, and by oral or nasal administration of protein or peptides.     

Current status of gene therapy for hemophilia

The hemophilias are an attractive model for gene therapy because their clinical manifestations are attributable to the lack of a single protein that circulates in minute amounts in the plasma. Sustained therapeutic expression of factors VIII and IX has been achieved in preclinical studies using a wide range of gene transfer technologies targeted at different tissues. This achievement has led to six different phase I/II clinical trials that resulted in limited efficacy but minimal toxicity. Recombinant adeno-associated viral vectors appear most promising for hemophilia gene therapy; however, this review summarizes all the major gene therapy approaches used and outlines the future challenges.     

An update on gene therapy for hemophilia

More than two thirds of the world's hemophiliacs currently face a drastically shortened life of pain and disability because they do not have access to safe factor concentrates, as these products are highly expensive and in limited supply. For these individuals, gene therapy remains an important avenue of hope because of its potential for a durable cure following a single therapeutic manipulation. The results of recent hemophilia gene clinical trials are encouraging, although they have failed to demonstrate sustained correction of the bleeding diathesis. Although many obstacles still remain, continuing technologic improvements have resulted in impressive advances in this field, which bodes well for patients with hemophilia and other genetic disorders.     

The common marmoset as a target preclinical primate model for cytokine and gene therapy studies

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.     

A novel plasma filtration therapy for hepatic failure: preclinical studies

There is a need to develop artificial means of liver replacement and/or assistance with the aim of either supporting patients with borderline functional liver cell mass until their liver regenerates, or until a donor liver becomes available for transplantation. Selective plasma filtration is a novel approach to blood purification therapy designed to reduce the level of circulating toxins of hepatic and renal failure, mediators of inflammation and inhibitors of hepatic regeneration. The results of preclinical studies indicate that treatment of pigs with experimentally-induced fulminant hepatic failure is safe and effective in extending survival time and arresting brain swelling. In addition, the amount of ammonia, aromatic amino acids, IL6, TNFalpha and C3a removed during the 6-h treatment in the present study was higher by 34% to 175% than the total plasma content of those substances at the start of therapy.     

Preclinical animal models for hemophilia gene therapy: predictive value and limitations

Hemophilia A and B are excellent candidate disorders for the application of somatic cell gene therapy. One of the major advantages in the preclinical development of hemophilia gene therapy strategies has been the availability of several animal models for both hemophilia A and B. These models recapitulate many of the phenotypic aspects of human hemophilia and have proven to be very informative in exploring the efficacy and safety of gene therapy. Considerable progress has been made in the design of gene therapy protocols, and over the last 5 years it has been shown that long-term phenotypic correction, with sustained therapeutic levels of factor VIII (FVIII) and factor IX (FIX), can be attained in FVIII- and FIX-deficient mice and dogs using various viral vector-mediated gene therapy approaches. These animal models also have elucidated potential complications of gene therapy protocols, including acute vector-associated toxicities and the induction of neutralizing antibodies to the FVIII and FIX transgene products. Nevertheless, although the preclinical paradigm of hemophilic mouse followed by hemophilic dog studies has proven to be extremely helpful in evaluating the efficacy and safety of potential clinical gene therapy protocols, several limitations to these animal models still exist. This review presents a summary of the animal models available for hemophilia gene therapy, and highlights the various strengths and weaknesses of these models.     

Development of lentiviral vectors for gene therapy for human diseases

Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy. (Blood. 2000;95:2499-2504)     

Nonviral gene therapy approaches to hemophilia

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.     

Coagulation factors with improved properties for hemophilia gene therapy

Hemophilias A and B are X-linked bleeding disorders that result in a qualitative or quantitative deficiency in coagulation factors VIII (FVIII) and IX (FIX), respectively. Affected patients experience significant morbidity as a result of repeated joint hemorrhages and subsequent arthropathy, and there is increased mortality related to life-threatening bleeding events. The mainstay of therapy is episodic or prophylactic infusions of plasma-derived or recombinant FVIII or FIX. However, gene transfer holds the promise of maintaining plasma levels of FVIII or FIX high enough to prevent the development of joint disease and reduce the risk of life-threatening bleeds or possibly even achieving normal plasma levels. Human gene therapy trials thus far have fallen short of this goal. This review summarizes the inherent limitations in expression of recombinant FVIII and the bioengineering strategies that are currently being explored for constructing novel recombinant FVIII molecules that have improved function. Current strategies for FVIII include increasing mRNA levels, improving secretion efficiency, increasing the rate of thrombin activation, stabilization of the activated form of FVIII, and strategies to prolong FVIII half-life in plasma by disrupting FVIII interaction with its clearance receptors. Strategies to improve the function of FIX include increasing the mRNA levels, reducing interaction with collagen IV, and increasing the specific activity. These novel molecules partnered with advances in gene transfer vector design and delivery may ultimately achieve persistent expression of FVIII and FIX, leading to an effective long-term treatment strategy for the hemophilias.     

Adoptive T-cell therapy for B-cell acute lymphoblastic leukemia: preclinical studies.

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8(+) and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti-leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.     

Factor IX variants improve gene therapy efficacy for hemophilia B

Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.     

Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated virus has recently shown promise. This article will briefly outline a novel gene-transfer approach using self-complementary adeno-associated viral vectors using hemophilia B as a target disorder. This approach is currently being evaluated in the clinic. We will provide an overview of the development of self-complementary adeno-associated virus vectors as well as preclinical and clinical data with this vector system.     

Gene therapy for hemophilia

PURPOSE OF REVIEW: This review will highlight the progress achieved in the past 2 years on using gene therapy to treat hemophilia in animals and humans. RECENT FINDINGS: There has been substantial progress in using gene therapy to treat animals with hemophilia. Novel approaches for hemophilia A in mice include expression of Factor VIII in blood cells or platelets derived from ex-vivo transduced hematopoietic stem cells, or in-vivo transfer of transposons expressing Factor VIII into endothelial cells or hepatocytes. Advances in large-animal models include the demonstration that neonatal administration of a retroviral vector expressing canine Factor VIII completely corrected hemophilia A in dogs, and that double-stranded adeno-associated virus vectors resulted in expression of Factor IX that is 28-fold that obtained using single-stranded adeno-associated virus vectors. In humans, one hemophilia B patient achieved 10% of normal activity after liver-directed gene therapy with a single-stranded adeno-associated virus vector expressing human Factor IX. Expression fell at 1 month, however, which was likely due to an immune response to the modified cells. SUMMARY: Gene therapy has been successful in a patient with hemophilia B, but expression was unstable due to an immune response. Abrogating immune responses is the next major hurdle for achieving long-lasting gene therapy.